The human adenovirus (Ad) has been exploited as a vector for gene delivery [Benihoud et al. (1999) Curr Opin Biotechnol 10:440-7; Brenner (1999) Blood 94:3965-7; Kochanek (1999) Hum. Gene. Ther. 10:2451-9]. Adenovirus is a common DNA virus that naturally infects the airway epithelia as well as other tissues in the body. The advantages of using adenovirus in gene delivery include the facts that its life cycle has been well characterized, its genome may be easily manipulated in the laboratory, and recombinant viruses are readily grown to high titers. In addition, adenovirus has a wide host cell range that includes non-dividing cells in vitro and in vivo. It is possible to achieve efficient gene expression in quiescent and differentiated cells. Finally, adenovirus is a relatively benign human virus that is associated with mild disease, and importantly is not associated with the development of any human malignancy.
However, several disadvantages exist for the use of adenovirus as a vector for long term gene transfer. First, it is evident from animal studies that adenovirus elicits an inflammatory response shortly after infection, and a subsequent cytotoxic T cell response directed against virus-infected cells [reviewed in Wold (1999) Human Press, Totowa, N.J.]. The result is immune clearance of virus-infected cells and extinction of expression of any foreign gene introduced by the recombinant viral vector. In the context of gene therapy in which repeated application of adenovirus-derived vectors may be required for continued treatment of certain diseases, the rapid immune response to adenovirus infection severely compromises the use of this system for long term gene therapy. It appears likely that the expression of adenovirus encoded proteins leads to immune recognition [Tripathy et al. (1996) Nat. Med. 2:545-50; Yang et al. (1996) J. Virol. 70:7209-12]. A second disadvantage is that the Ad has no direct means to persist in infected cells [Benihoud et al. (1999) Curr Opin Biotechnol 10:440-7; Brenner (1999) Blood 94:3965-7; Kochanek (1999) Hum. Gene. Ther. 10:2451-9], thus further limiting its use for long term gene therapy.
To avoid some of the problems associated with using adenovirus in gene transfer, one approach of the prior art has been to generate “gutted” adenoviruses which lack all adenovirus coding regions. While gutted adenoviruses have the advantage of allowing efficient gene transfer as well as minimizing an adverse immune response, they nonetheless require serial passage, and stuffer fragments to maintain a certain genome size which allows for efficient propagation. Additionally, gutted adenoviruses are not stably integrated into the cell genome, thus limiting their use for long term gene transfer applications.
An alternative approach by the prior art to circumvent some of the limitations of adenovirus-based vectors has been to use adenovirus “hybrid” viruses which incorporate desirable features from adenovirus as well as from other types of viruses as a means of generating unique vectors with highly specialized properties. For example, viral vector chimeras were generated between adenovirus and adeno-associated virus (AAV) [Thrasher et al. (1995) Gene Ther. 2:481-485; Fisher et al. (1996) Hum. Gene Ther. 7:2079-2087; Lieber et al. (1999) J. Virol. 73:9314-9324; Liu et al. (1999) Gene Ther. 6:293-299]. However, generation of the adenovirus/adeno-associated virus vectors of the prior art is inefficient.
Thus, what is needed are compositions and methods for efficient generation of vectors that may be used in gene transfer applications which are exemplified by, but not limited to, gene therapy applications. Preferably, these compositions and methods should also be non-immunogenic and non-toxic, and should permit stable integration into cells.